This application is a 371 of PCT/GB99/00238 filed Jan. 22, 1998.
The present invention relates to methods of preparing hybrid seed.
In particular, the present invention relates to the molecular control of sterility in crop plants. Such male and female sterility in plants can be used in the preparation of hybrid seed from crops which are naturally self-pollinators.
The present invention also provides for a method of restoring fertility in the parent plants to allow self-pollination, thereby allowing the maintenance of the parental lines.
The present invention further relates to expression cassettes for incorporation into plants and to the use of such expression cassettes in a male/female sterility restorer system.
Hybrid plants grown from hybrid seed benefit from the heterotic effects of crossing two distinct genetic backgrounds. The production of hybrid seed depends on the ability to control self-pollination and ensure cross-pollination of male and female parent plants.
A number of methods are available to control pollen fertility. For example, in the case of maize, which has separate male and female flowers, control of pollen fertility is achieved by physically removing the male inflorescence or tassel, prior to pollen shed, thus preventing self-pollination.
Most major crops, however, have both functional male and female reproductive organs within the same flower. In this instance, removal of the pollen producing organs is very labour intensive and expensive. The use of chemicals (gametocides), particularly in wheat, maize (corn) and rice, to kill or block pollen production produces transitory male sterility but the use of such chemicals is expensive. The reliability of the chemicals and their length of action are also issues.
There is considerable interest in developing systems of pollen control based on genetic mechanisms producing male sterility. There are two general types: a) nuclear male sterility caused by the failure of pollen production due to one or more nuclear genes and b) cytoplasmic male sterility (CMS) in which pollen production is blocked because of a defect in a gene in the mitochondria.
Currently available nuclear systems are based on the introduction of a male sterility trait to one parent plant followed by the introduction of a fertility restoration gene as a result of cross-pollination with another plant to produce fertile hybrid plants. The Paladin system, which is described in WO96/01799, is different and is based on the separation during hybrid seed production of genes which, when expressed together in one plant, have a cytotoxic effect leading to male sterility.
Rice and wheat are self-pollinating plants and have small hermaphrodite flowers and so the detasseling approach taken for hybrid seed production in maize is not applicable. Manual removal of anthers is difficult and time consuming. Moreover, wheat pollen is relatively heavy and is viable only for a short time, rarely remaining viable for longer than 30 minutes. The technique of planting used in hybrid corn production i.e. planting the male parent in a block physically separated from the female parent (the male sterile) and allowing wind pollination does not, therefore, work well in wheat or rice. The male and female parents for these crops have to be interplanted to ensure cross pollination. As hybrid seed needs to comprise more than 95% hybrids, it is necessary to remove seed arising from self-pollination of the male parent or to make the male parent incapable of self-fertilisation and therefore incapable of producing non-hybrid seed. Clearly, the interplanting of the parent plants means that the first option is difficult unless the male plants are susceptible to some chemical treatment to which the female parent is tolerant e.g. herbicide treatment.
Our International Patent Application No. PCT/GB90/00110 describes a cascade of gene sequences which expresses a protein which disrupts the biosynthesis of viable pollen in a female parent plant. In this case, however, only one of the parent plants i.e. the female parent is sterile to minimise self-pollination of the female plant and this female plant is crossed with a fertile male parent plant to yield fertile hybrid seed. There is no description in the literature, however, of a method of producing hybrid seed wherein both parent plants are unable to self-pollinate.
The present invention relates to two methods by which hybrid seed may be produced which seeks to overcome the problems presently associated with the production of hybrid seed, particularly with the production of hybrid wheat and rice seed.
According to a first aspect of the present invention, there is provided a method of preparing hybrid seed comprising interplanting a male parent plant which is male fertile and homozygous recessive female sterile and a female parent plant which is homozygous recessive male sterile and female fertile, allowing cross-pollination and obtaining seed produced therefrom.
According to a second aspect of the present invention, there is provided the use of the above method to produce hybrid seed.
According to a third aspect of the present invention, there is provided fertile plants produced by the aforementioned method.
According to a fourth aspect of the present invention there is provided the progeny of the aforementioned plants, the seeds of such plants and such progeny.
According to a fifth aspect of the present invention there is provided an expression cassette comprising:
(a) a first gene promoter sequence which is a male flower specific promoter sequence;
(b) a disrupter gene encoding a product capable of disrupting male fertility operably linked to the first gene promoter sequence;
(c) a second gene promoter sequence which is a female flower specific promoter sequence optionally operably linked to one or more translational enhancer or intron sequences;
(d) a restorer gene encoding a product capable of restoring female fertility operably linked to the second gene promoter sequence;
(e) a third gene promoter sequence responsive to the presence of an exogenous chemical inducer optionally operably linked to one or more translational enhancer or intron sequences; and
(f) a restorer gene encoding a product capable of restoring male fertility operably linked to the third gene promoter sequence;
whereby the presence of the exogenous chemical inducer controls male fertility.
According to a sixth aspect of the present invention there is provided an expression cassette comprising:
(a) a first gene promoter sequence which is a female flower specific promoter sequence;
(b) a disrupter gene encoding a product capable of disrupting female fertility;
(c) a second gene promoter sequence which is a male flower specific promoter sequence optionally operably linked to one or more translational enhancer or intron sequences;
(d) a restorer gene encoding a product capable of restoring male fertility operably linked to the second gene promoter sequence;
(e) a third gene promoter sequence responsive to the presence or absence of an exogenous chemical inducer optionally operably linked to one or more translational enhancer or intron sequences; and
(f) a restorer gene encoding a product capable of restoring female sterility operably linked to the third gene promoter sequence;
whereby the presence of the exogenous chemical inducer controls female fertility.
According to a seventh aspect of the present invention there is provided a further method of producing hybrid seed comprising incorporating a first expression system according to the fifth aspect of the present invention into a first plant to generate a hemizygous female parent plant and incorporating a second expression system according to the sixth aspect of the present invention into a second plant to generate a hemizygous male parent plant;
applying an exogenous chemical inducer to the transformants thereby allowing the plants to self-pollinate;
growing up plants from the resulting seed;
selecting for male and female homozygous plants;
crossing the selected male and female plants; and
obtaining the resulting hybrid seed.
According to an eighth aspect of the present invention there is provided plant tissue transformed with either one of the expression cassettes as defined above and material derived from the said transformed plant tissue.
According to a ninth aspect of the present invention there is provided fertile whole plants comprising the tissue or material as defined above.
According to a tenth aspect of the present invention there is provided the progeny of the selected plants produced according to the seventh aspect of the present invention, the progeny comprising expression cassettes as defined above incorporated, preferably stably incorporated, into their genome and the seeds of such plants and such progeny.
According to an eleventh aspect of the present invention, there is provided a plant, the genome of which comprises the first expression cassette according to the fifth aspect of the present invention.
According to a twelfth aspect of the present invention, there is provided a plant, the genome of which comprises the second expression cassette according to the sixth aspect of the present invention.
According to a thirteenth aspect of the present invention, there is provided hybrid seed produced by crossing these two plants and obtaining the resulting hybrid seed produced therefrom.
According to an fourteenth aspect of the present invention there is provided the use of the second method according to the present invention to produce hybrid seed.
According to a fifteenth aspect of the present invention there is provided a method of transforming a plant comprising incorporating into the genome of the plant an expression cassette as defined above wherein the restorer gene, which is operably linked to a third gene promoter sequence, is inducibly expressed in the target tissue but may be constitutively expressed in one or more other tissues so that the disrupter gene is only effective in the target tissue. The third promoter sequence may be constitutively expressed at a particular stage e.g. in callus tissue.
Preferably, the first method of the present invention wherein the genomic DNA of each parent plant has integrated therein a gene construct comprising a promoter sequence responsive to the presence or absence of an exogenous chemical inducer, optionally operably linked to one or more translational enhancer or intron sequences, operably linked to a gene which fully restores the fertility of each parent plant, the gene being expressed by the application to the plant of an external chemical inducer thereby allowing each parent to self-pollinate.
Preferably, the female parent plant is homozygous for a recessive gene which disrupts the biogenesis of viable pollen or which significantly reduces the viability of the pollen.
Preferably, the male parent plant is homozygous for a recessive gene which disrupts female floral structures such as ovule, style, stigma in such a way that fertilisation is prevented, or adhesion, hydration or germination of pollen inhibited or which inhibits pollen tube growth or guidance.
Preferably, the inducible promoter sequence is the AlcA promoter sequence or the GST-27 promoter sequence.
Preferably, the parent plants are wheat, barley, rice, maize, sugarbeet, tomato, sunflower, canola, cotton, soybean and other vegetables such as lettuce.
Preferably, the F1 hybrid seed produced by the first method of the present invention gives rise to plants, all of which are fully fertile.
Preferably, the F2 hybrid seed produced by the first method of the present invention gives rise to plants which segregate for sterility, about 25% being female sterile.
Preferably, the sterility of the parents is caused by a natural or genetically manipulated mutation.
Preferably, the first expression cassette defined above and used in the second method of the present invention comprises a disrupter gene encoding a product which is capable of disrupting pollen production.
Preferably, the first expression cassette defined above comprises a disrupter gene encoding a product which is capable of being expressed in the tapetal cells of the plant.
Preferably, the third gene promoter sequence in the first expression cassette is the AlcA promoter sequence or the GST-27 promoter sequence.
Preferably, the second expression cassette defined above and used in the second method of the present invention comprises a restorer gene encoding a product which is capable of restoring pollen production.
Preferably, the second expression cassette defined above comprises a restorer gene which is capable of overcoming disruption of the tapetal cells.
Preferably, the third gene promoter sequence in the second expression cassette is the AlcA promoter sequence or the GST-27 promoter sequence.
Preferably, the male plants in the second method according to the present invention comprise a homozygous dominant gene restoring male fertility.
Preferably, the female plants in the second method according to the present invention comprise a homozygous dominant gene restoring female fertility.
Preferably, the F1 hybrid seed produced gives rise to plants, the anthers of which produce approximately 50% of viable pollen, where the first gene promoter sequence of the first expression cassette is a gametophytic promoter sequence.
Preferably, the F1 hybrid seed produced gives rise to plants, all of which are fully fertile where the first promoter sequence of the first expression cassette is a sporophytic promoter sequence.
Preferably, the F2 hybrid seed gives rise to plants which segregate for sterility, of which a significant number are female sterile.
Preferably, the male and female homozygous plants produced by the second method according to the present invention are multiplied and maintained by the application of an exogenous chemical inducer to the plants, thereby allowing the plants to self-pollinate. In this regard, further generations of self-pollination of the selected male and female homozygous plants can be produced and when hybrid seed is required, the plants may be crossed to obtain hybrid seed.
Preferably, the plants used in the second method of the present invention are wheat, barley, rice, maize, sugarbeet, tomato, sunflower, canola, cotton, soybean and other vegetables.
Preferably, the restorer gene used in the method of transforming a plant according to the present invention is constitutively expressed in callus tissue from which transformed plants are regenerated.
Preferably, the restorer gene is inducibly expressed in the male or female flower structures.
Preferably, the third gene promoter sequence of the expression cassettes used in the transformation process is the GST-27 or the AlcA promoter sequence.
A preferred embodiment of the present invention is a method of preparing hybrid seed comprising interplanting a male parent plant which is male fertile and homozygous recessive female sterile and a female parent plant which is homozygous recessive male sterile and female fertile, allowing cross-pollination and obtaining seed produced therefrom wherein the genomic DNA of each parent plant has integrated therein a gene construct comprising a promoter sequence responsive to the presence or absence of an exogenous chemical inducer operably linked to a gene which fully restores the fertility of each parent plant, the gene being expressed by the application to the plant of an external chemical inducer thereby allowing each parent to self-pollinate when required for multiplication of the seed stocks of each parent plant.
A further preferred embodiment of the present invention is an expression system comprising:
(a) a first gene promoter sequence which is a male flower specific promoter sequence;
(b) a disrupter gene encoding a product capable of disrupting male fertility operably linked to the first gene promoter sequence;
(c) a second gene promoter sequence which is a female tissue specific promoter sequence optionally operably linked to one or more translational enhancer or intron sequences;
(d) a restorer gene encoding a product capable of restoring female fertility operably linked to the second gene promoter sequence;
(e) a third gene promoter sequence responsive to the presence or absence of an exogenous chemical inducer optionally operably linked to one or more translational enhancer or intron sequences;
f) a restorer gene encoding a product capable of restoring male fertility operably linked to the third gene promoter sequence;
whereby the presence of the exogenous chemical inducer controls male fertility, wherein the gene capable of disrupting male sterility is a disrupter gene encoding a product which is expressed in the tapetal cells of the plant.
Another preferred embodiment of the present invention is an expression system comprising:
(a) a first gene promoter sequence which is a female tissue specific promoter sequence;
(b) a disrupter gene encoding a product capable of disrupting female fertility;
(c) a second gene promoter sequence which is a male tissue specific promoter sequence optionally operably linked to one or more translational enhancer or intron sequences;
(d) a restorer gene encoding a product capable of restoring male fertility operably linked to the second gene promoter sequence;
(e) a third gene promoter sequence responsive to the presence or absence of an exogenous chemical inducer optionally linked to one or more translational enhancer or intron sequences; and
(f) a restorer gene encoding a product capable of restoring female fertility operably linked to the third gene promoter sequence;
whereby the presence of the exogenous chemical inducer controls female fertility and wherein the gene capable of restoring male fertility is a gene which encodes a product which restores pollen production in the tapetal cells.
The preferred male flower specific promoter sequences are the maize MSF14 and C5 (derived from pectin methyl esterase) promoter sequences.
The term xe2x80x9cplant materialxe2x80x9d includes a developing caryopsis, a germinating caryopsis or grain, or a seedling, a plantlet or plant, or tissues or cells thereof, such as the cells of a developing caryopsis or the tissues of a germinating seedling or developing grain or plant (eg in the roots, leaves and stem).
The term xe2x80x9ccassettexe2x80x9d which is synonymous with terms such as xe2x80x9cconstructxe2x80x9d, xe2x80x9chybridxe2x80x9d and xe2x80x9cconjugatexe2x80x9d includes a gene of interest directly or indirectly attached to a gene promoter sequence. An example of an indirect attachment is the provision of a suitable spacer group such as an intron or enhancer sequence intermediate the promoter and the gene of interest. Such constructs also include plasmids and phage which are suitable for transforming a cell of interest.
The term xe2x80x9cdisrupter genexe2x80x9d is a gene which acts in a dominant fashion, and when expressed at a suitable stage of plant development, will lead to the failure of a plant to form normally functioning female flower structures or normally functioning male flower structures so that the plant is female or male sterile. Such a gene may exert its effect by disrupting tissues such as the tapetum and endothelium. The gene may be expressed specifically in male flowers during pollen formation causing cell death of the anthers and associated tissues, pollen mother cells, pollen and associated tissues. It may also be expressed in the stigma or in the transmitting tract of the style, thus interfering with the process of pollen adhesion, hydration, pollen germination and pollen tube growth and guidance. The origin of the disrupter genes can be from a variety of naturally occurring sources e.g. human cells, bacterial cells, yeast cells, plant cells, fungal cells, or they can be totally synthetic genes which may be composed of DNA sequences, some of which may be found in nature, some of which are not normally found in nature or a mixture of both. These genes will preferably have an effect on mitochondrial metabolism, as it is known that a good energy supply is an absolute requirement for the production of fertile pollen. The disrupter genes may, however, be effectively targeted to other essential biochemical functions such as DNA and RNA metabolism, protein synthesis, and other metabolic pathways. The preferred dominant disrupter gene is barnase.
The term xe2x80x9crestorer genexe2x80x9d is a gene which acts in a dominant fashion, and when expressed, will reverse the effects of the disrupter gene. The preferred dominant restorer gene is barstar.
The term xe2x80x9cfemale flowerxe2x80x9d is intended to include all parts of the female reproductive organs including but not limited to, ovary, ova, pistil, style, stigma, transmitting tract, placenta.
The term xe2x80x9cmale flowerxe2x80x9d is intended to include all parts of the male flower, including but not limited to, the tapetum, anthers, stamens, pollen.
The methods of hybrid seed production according to the present invention are different from and have a number of advantages over existing methods in a number of ways. The utilisation of both male and female sterility has not previously been described. This feature prevents self pollination of either parent thus allowing the production of hybrid seed without the need for separate planting blocks for male and female parents. This interplanting of male and female parent plants maximises the opportunity for cross pollination in crops, such as wheat and rice, which are essentially self pollinators. In the examples of wheat and rice, where block planting is not carried out, this method allows production of hybrid seed without the need to apply herbicide to rogue out male parent plants after fertilisation of the female parent. A chemically inducible restorer system is needed only for the maintenance of homozygous parental lines rather than for the hybrid seed production process. This means that chemicals are applied to limited acreages and then only infrequently. A number of disrupter-restorer systems, or operator-repressor systems may be used in the present invention.
Plants containing the expression cassettes of the present invention which control male and female fertility may also be used separately to make F1 hybrids with other parent lines, which do not contain the expression cassettes, if suitable alternative control of male or female fertility (such as mechanical removal of anthers or ovules, or use of chemical gametocides) is used in the other line. If the progeny from these F1 hybrids are then backcrossed for an appropriate number of generations to the other hybrid parents, whilst selecting for the presence of the expression cassette with molecular, biochemical or progeny-testing techniques, the system for controlling male or female fertility can be transferred or introgressed into new parent backgrounds. Alternatively, F1 hybrids with other parent lines can be self-pollinated, through application of an exogenous chemical inducer to restore male or female fertility as appropriate, so as to select new hybrid parents containing the expression cassettes, through the normal process of plant breeding. Use of such introgression and plant breeding will permit the methods of hybrid seed production of the present invention to be used with a wide variety of new and existing F1 hybrid parental combinations.
Promoters which are inducible by application of exogenous chemicals are known in the art. Suitable inducible promoters are those which are activated by application of a chemical, such as a herbicide safener. Examples of inducible promoters include AlcA/R switch system described in our International Publication No. WO. 93/21334, the GST switch system described in described in International Publication Nos WO 90/08826 and WO 93/031294 or the ecdysone switch described in International Publication No. WO 96/37609. Such promoter systems are herein referred to as xe2x80x9cswitch promotersxe2x80x9d. The switch chemicals used in conjunction with the switch promoters are agriculturally acceptable chemicals making these promoters particularly useful in the methods of the present invention.
One of the advantages of using the AlcA promoter, which is a component of the Alc A/R switch system, in the present invention is that the chemical inducer used is ethanol. This chemical is advantageous in that it can be applied as a root drench, as an aqueous spray, or as a gas. It is effective at concentrations of 1% and is non-toxic to operators and to the environment.
The present invention can be used for any mono- or di-cotyledonous plant which the breeder or grower wants to produce as F1 hybrid seed and for which suitable transformation techniques are or become available, particularly wheat and rice crops. The present invention has the advantage of reducing crop management costs associated with the F1 hybrid seed production, ease of purity control of hybrid seed and maintenance of parental lines.
In a particular application, the present invention relates to the production of male and female parental plants, which are rendered sterile using molecular engineering techniques. The sterility of these plants can be reversed by using a chemical application which leads to the restoration of fertility.
The anther is the site of male reproductive processes in flowering plants. It is composed of several tissues and cell types and is responsible for producing pollen grains that contain the sperm cells. The tapetum is a specialised tissue which plays a critical role in pollen formation. It surrounds the pollen sac early in pollen development, degenerates during the latter stages of development and is not present in an organised form in the mature anther. The tapetum produces a number of compounds which aid pollen development or are incorporated into the pollen outer wall and it has been demonstrated that many of the natural male sterility mutations have impaired tapetum differentiation or function. Tapetal tissue is therefore critical to the formation of functional pollen grains.
A number of genes have been identified and cloned that are specifically expressed in tapetal tissue. They include Osg6B, Osg4B (Tsuchiya et al. 1994, Yokoi, S et al. 1997), pE1, p T72 (WO9213957), p CA55 corn (WO92/13956), TA29, TA13, (Seurinck et al 1990), RST2 corn (W09713401), MS14,18,10 and A6, A9 from Brassica napus (Hird et al. 1993).
A tapetum specific promoter isolated from rice has been shown to give rise to male sterile plants when used to drive expression of xcex2 1,3 glucanase in tobacco, (Tsuchiya et al. 5 1995). The tapetum specific promoter TA29 has been used to produce male sterile tobacco (Mariani et al 1990) and pCA55, pE1 and pT72 to produce male sterile wheat (De Block et al. 1997) when driving the expression of barnase.
Pollen specific clones have been obtained from a number of species, including corn (Hanson et al. 1989, Hamilton et al. 1989,) and tomato (Twell et al. 1990, 1991).
Anther specific clones have been isolated from a number of species Bp4A and C from Brassica napus (Albani et al. 1990), chs from petunia (Koes et al. 1989), rice (Xu et al. 1993, Zou et al. 1994), amongst others.
Wheat homologues of these clones and others may be obtained by such methods as degenerate PCR, utilising sequences found in the literature, and subsequent screening of wheat and other genomic libraries, and analysis of tissue specificity using the expression of reporter genes. These methods are well documented in the literature.
In higher plants the female reproductive organ is represented by the pistil, composed of the ovary, style and stigma. The gynoecium has been shown to contain up to 10,000 different mRNAs not present in other organs (Kamalay and Goldberg 1980). These include regulatory genes responsible for controlling pistil development as well as xe2x80x9cdownstreamxe2x80x9d ones encoding proteins associated with differentiated cell types in the pistil. Genes governing self-incompatibility and their homologues are one class of gene with pistil predominant expression patterns (Nasrallah et al. 1993). Other cloned genes include xcex2 glucanase (Ori et al. 1990), pectate lyase (Budelier et al. 1990) and chitinase (Lotan et al. 1989) which are expressed in the transmitting tissue and a proteinase inhibitor (Atkinson et al. 1993) which are expressed in the style. Others are pathogenesis related or are homologues of genes involved in the cleavage of glycosidic bonds. These enzymes may facilitate pollen tube growth by digesting proteins in the tissue through which the pollen tube grows.
A number of female sterile mutants have been identified in Arabidopsis. For example, sin1 (short integument) (Robinson-Beers et al. 1992) and bel1 (bell) (Robinson-Beers et al. 1992) affect ovule development. The Aintegumenta mutation blocks megasporogenesis at the tetrad stage (Elliot, R. C, et al. 1996, Klucher, K. M, 1996). A lethal ovule 2 mutation has been observed but not cloned in maize (Nelson et al. 1952). Pistil specific basic endochitinases have been cloned from a number of species (Ficker et al. 1997, Dzelzkalns et al. 1993, Harikrishna et al. 1996, Wemmer et al. 1994) and extensin-like genes have been shown to be expressed in the styles of Nicotiana alata (Chen C-G, et al. 1992).
The following are ovule specific clones ZmOV23,13, (Greco R., et al. unpublished), OsOsMAB3A (Kang H. G., et al. 1995), ZmZmM2 (Theissen G., et al. 1995) and stigma specific stig1 (Goldman, M. H et al. 1994), STG08, STG4B12 (EP-412006-A). Goldman et al. used the promoter from the STIG1 gene to drive expression of barnase in the stigmatic secretory zone. This led to flowers having no secretory zone in the pistils and thus were female sterile. Pollen grains were able to germinate but were unable to penetrate the surface.
Seven ovule specific cDNAs have been isolated from orchid (Nadeau et al. 1996). Again, wheat homologues of these and any others may be obtained by standard molecular biology techniques.
Another aspect to the methods of the present invention is the identification of genes impacting on male and female sterility. Such genes can be used in a variety of systems to control fertility.
The procedure for tagging maize genes with transposable elements has been reviewed (Doring, 1989). One of the methods which can be used is to cross a maize line carrying active transposable elements and a dominant allele of the target gene with a normal maize strain that does not carry transposable elements. Progeny from the cross can be selfed and screened for the most desirable mutations, i.e. those that lead to sterility. The sterile plants represent potential instances in which a transposable element has transposed to a locus bearing a gene essential for fertility. The genes may then be recovered in a variety of ways. U.S. Pat. No. 5,478,369 describes the isolation by this method of a gene described as MS45.
A male fertility gene has been identified in Arabidopsis thaliana using the En/Spm-I/dSpm transposon tagging system to obtain a male sterility 2 (ms2) mutant and the MS2 gene (Aarts et al. 1993). This MS2 gene has been shown to be involved in male gametogenesis, cell wall synthesis does not proceed after microspore mother cell meiosis and the microspores are eventually degraded. Homologues of MS2 have been identified in Brassica napus, Zea Mays and to an open reading frame found in wheat mitochondrial DNA. The isolation of genes critical to fertility in Arabidopsis may therefore lead to the cloning of homologues in other species. This approach can clearly be taken to isolate other genes critical to fertility.
There is now evidence for the existence of extensive regions of conserved colinearity among grass species at the genetic level. Ahn and Tanksley (1993) showed the relationship between rice and maize and Kurata et al (1994) showed that the wheat genome could be aligned with rice and Moore et al (1995) showed that all three maps could be aligned. This opens the way to the use of comparative genome mapping as a means of gene isolation.
The microsynteny approach to gene cloning is based on the emerging similarity in molecular marker and gene order among evolutionary related species. This approach is particularly attractive for large genome cereal species of agricultural importance like wheat, maize and barley that may take advantage of their small genome relative, rice. Kilian et al. (1997) report on progress on map-based cloning of the barley RpgI and rpg4 genes using rice as an intergenomic mapping vehicle.
As the above approach is limited to target genes which have been genetically mapped, an alternative method of gene isolation, which is an effective transposon tagging system, is being developed in rice using the maize Ac/Ds system, Izawa et al (1997).
A number of methods have been suggested as being useful to inactivate genes necessary for fertility or to produce cytotoxic compounds in the tissues to prevent normal development of gametophytes.
Our International patent application no. PCT/GB96/01675 describes a method of inhibiting gene expression in a target plant tissue using a disrupter gene selected from zANT, tubulins, T-urf, ATP-ase subunits, cdc25, ROA, MOT.
There are several other known inactivating systems. For example, barnase (Mariani et al. 1990), diphtheria toxin A-chain, pectate lyase. Two examples of expressing cytotoxic compounds previously described are avidin expression and IamH/IamS,
The expression of xcex2-1,3-glucanase in tapetal cells has been shown to generate male sterile plants (Worrall et al. 1992). Anti-sense has been proposed as a mechanism by which the expression of genes critical to pollen development can be down regulated and it has been shown (Van der Meer 1992) that antisense inhibition of flavonoid biosynthesis does indeed lead to male sterility. Reduction of flavanol expression has been claimed in maize to result in male sterility (WO 93 18171 Pioneer Hi-Bred International). Other mechanisms have also been described (Spena et al. 1992).
Baulcombe (1997) describes a method of gene silencing in transgenic plants via the use of replicable viral RNA vectors (Amplicons(trademark)) which may also be useful as a means of knocking out expression of endogenous genes. This method has the advantage that it produces a dominant mutation i.e. is scorable in the heterozygous state and knocks out all copies of a targeted gene and may also knock out isoforms. This is a clear advantage in wheat which is hexaploid. Fertility could then be restored by using an inducible promoter to drive the expression of a functional copy of the knocked out gene.
Kempin et al. (1997) report the targeted disruption of a functional gene using homologous recombination. This method normally, however, produces a recessive mutation i.e. is scorable only in the homozygote. To be detectable in the heterozygous state it would either have to be lethal directly or, for example, cause a block in a pathway such that there was a build up of a cytotoxic compound leading to lethality. In order to detect a sterility mutation it would be necessary to generate a recessive homozygote by self-pollination, where one in four of the progeny would be sterile. The switch construct allowing expression of the knocked out gene would have to be introduced into a heterozygote obtained by back crossing the homozygote. This is a potentially useful method for generating the recessive mutants needed for the method later described in Example 1.
Ribozymes are RNA molecules capable of catalysing endonucleoolytic cleavage reactions. They can catalyse reactions in trans and can be targeted to different sequences and therefore are potential alternatives to antisense as a means of modulating gene expression. (Hasselhof and Gerlach). Wegener et al (1994) have demonstrated the generation of a trans-dominant mutation by expression of a ribozyme gene in plants.
Several methods are known for altering plant self incompatibilty systems by modifying S-gene expression as a means of introducing male sterility in which a plant is transformed with a construct utilising a gametophytic S-gene encoding a ribonuclease in such a way that a self-incompatible plant is converted to self compatible or that a self-compatible plant is converted to self-incompatibility, thus preventing self pollination.
Examples of combinations of disrupter/restorer genes include barnase and barstar, and TPP and TPS. The use of the barnase/barstar system to firstly generate sterility and then restore fertility has been described (Mariani et al. (1992). Trehalose phosphate phosphatase (TPP) when expressed in tapetal cells of tobacco using the tapetum specific promoter Tap1 (Nacken et al 1991) from Antirrhinum results in male sterility. It is thought to be as a result of changing the carbohydrate metabolic and photosynthetic capacity of the tissues in which it is expressed. Anthers show signs of necrosis and any pollen produced is dead. Back crossing with wild type tobacco results in normal seed development. Analysis of the progeny shows that sterility segregates with the transgene. TreC, trehalose-6-phosphate hydrolase is a second gene whose expression perturbs of levels of trehalose-6-phosphate and it has been shown that when it is expressed using the constitutive plastocyanin promoter the result is bud excision before flowering. Thus, if expression is limited to the tapetum, male sterility may result in the same way as when TPP is expressed in the tapetum. It has also been shown that after GA application flower buds remain on the plant and some pollen is produced leading in some cases to seed production.
It has also been shown that simultaneous equimolar expression of trehalose phosphate synthase (TPS) and TPP gives no effect on plant physiology i.e. TPS counteracts the effect of TPP on carbohydrate metabolic and photosynthetic capacity of tissues in which they are expressed. It has also been shown that it is possible to restore fertility by retransforming sterile tobacco lines with a construct expressing TPS in the tapetun. Clearly, expression of TPS could also be put under control of an inducible promoter to allow fertility to be restored when desired, or optimising GA application could be an alternative means of restoring fertility. The promoter from a gene expressed specifically in the tissues surrounding or in, the ovule, such as the MADS box gene FBP7 could be used to drive expression of TPP or TreC to obtain female sterility. It is likely that some optimisation of codon usage may be required to obtain the same effect in a monocot crop plant such as wheat or corn, (Merlo and Folkerts), (Seed and Haas).
The use of a number of operator/repressor systems has been described as a means of controlling gene expression in plants. Wilde et al. demonstrated the use of the E. coli lac system to repress expression of GUS under the control of the maize cab promoter (lac I expression driven by 35S CaMV promoter). Operator sequences were inserted at various positions within the CAB promoter and the extent of repression assessed. Depending upon the position of the operator sequences, a range of repression was observed. When the operator sequence was incorporated by replacement between the TATA box and the transcription start, repression of xcx9c90% was obtained. This repression can be relieved by the addition of IPTG. This was shown both in tobacco protoplasts and stable transformants.
Lehming et al. report that dramatic changes in binding affinity may be achieved by the modification of amino acids in the recognition helix of the lac repressor thus giving a tighter control of expression. Other such systems have been described and include the tetracycline inducible promoter system developed by Gatz et al (1991, 1992) in which a modified 35S CaMV promoter is repressed in plants expressing high levels of the tetracycline repressor protein but restored when tetracycline is added.
Steroid induction of protein activity can provide a chemically inducible expression system which does not suffer from chemical toxicity problems. Ligand binding domains of mammalian and insect steroid receptors such as glucocorticoid receptor (GR), oestrogen receptor can be used to regulate the activity of proteins in mammalian cells (Picard et al. 1993). A ligand binding domain fused to a protein maintains the protein in an inactive state until the ligand is introduced. Lloyd et al. (1994) describes a fusion of a maize transcriptional regulator with GR. Simon et al. (1996) describe a fusion of GR with an Arabidopsis flowering time gene product responsible for induction of transcription and Aoyama et al. (1995) describe a fusion of Ga14 or VP16 with a plant transactivating protein, Athb-1, placed under steroid control by means of the GR ligand-binding domain. It is known that the ability of transcriptional activators to bind to DNA and to simultaneously activate transcription is localised in defined domains of such transcription factors. It has been demonstrated (Ptashne 1988) and Mitchell and Tijan (1989) that transcriptional activator factors are made up of independently finctioning modules. Ptashne and Gann (1980) and others have shown that it is possible to combine a portion responsible for transcription activation of one factor with a DNA binding portion of another factor and the resulting hybrid protein be active in yeast cells. A system incorporating these components may be used to relieve repression and thus induce expression of genes in a controlled manner.
The use of juvenile hormone or one of its agonists as a chemical ligand to control gene expression in plants by receptor mediated transactivation has also been described.
Preferably, the switch system used to inducibly express the restorer gene is the AlcA/R switch system. We have demonstrated inducible expression of GUS in tomato anthers and pollen and have introduced similar constructs into wheat to demonstrate male and female tissue GUS expression using the AlcA/R switch system. We have also demonstrated inducible GUS expression in maize tassels, silks, embryo and endospermn using the safener inducible GST switch system (Jepson et al. 1994). Other switch systems may of course also be useful in the present invention.
The expression of cytotoxic or disrupter genes during plant transformation as a result of xe2x80x9cleakyxe2x80x9d expression from the male and female flower specific promoters at a very low level in tissues other than the target tissue may cause cell death and no recovery of transformants.
The inducible promoters used in the present invention to drive expression of the restorer genes may offer some protection against this possibility for the following reasons.
In the case of the GST-27 promoter, constitutive expression has been observed in callus. In the case of the AlcA promoter which is induced with ethanol, induction of GUS expression has been observed at concentrations of 7 ng/100 ml air. This concentration of ethanol can be added to the tissue culture medium to ensure expression of the restorer gene.
Examples of combinations of disrupter/restorer genes include barnase and barstar, and TPP and TPS.
The use of translational enhancer sequences, in particular the TMV xcexa9 sequence (Gallie et al.) is preferred in the present invention to give an increase in expression levels from the constitutive tissue specific and inducible promoters such that the expression of the restorer gene e.g. barstar, is far in excess of that needed to inhibit the disrupter gene e.g. barnase being produced. Gallie et al. showed that the translation of prokaryotic and eucaryotic mRNA""s is greatly enhanced by a contiguous derivative of the 68 nucleotide, 5xe2x80x2 leader sequence of tobacco mosaic virus U1 strain called xcexa9. Several other viral leader sequences have also been shown to enhance expression, such as alfalfa mosaic virus (A1MV) and broom mosaic virus (BMV). In tobacco mesophyll protoplasts an enhancement of xcx9c20 fold was observed. Other enhancer sequences e.g. tobacco etch virus may also be used in the present invention.
In addition, the use of intron sequences to enhance expression levels is well documented. Among those studied are the maize adh 1 intron 1 sequence which has been shown to increase levels of expression 12-20 fold when inserted in 5xe2x80x2 translated sequences in chimeric constructs introduced into maize protoplasts (Mascarenhas et al. 1990) and the Sh 1 intron also from maize. The inclusion of this intron into constructs in which the CaMV35S promoter was driving CAT expression resulted in increases of between 11 and 90 fold. (Vasil et al 1989).
Expression levels of the restorer and disrupter genes can also be balanced or modulated in the following ways. A promoter giving high levels of expression could be used to drive expression of the restorer gene while a promoter giving lower levels of expression could be used to drive expression of the disrupter gene. This would ensure that the disrupter gene product is swamped by restorer gene product thereby inactivating all cytotoxic or disrupter molecules allowing full restoration of fertility. A further way of modulating expression levels could be carried out by using mutagenesis to change the sequence around the AUG initiation codon in such a way that expression of the disrupter gene is non-optimal (Kozak (1989)) and is therefore down-regulated.
The expression systems of the present invention can be introduced into a plant or plant cell via any of the available methods such as Agrobacterium transformation, electroporation, microinjection of plant cells and protoplasts, microprojectile bombardment, bacterial bombardment, particularly the xe2x80x9cfibrexe2x80x9d or xe2x80x9cwhiskerxe2x80x9d method, depending upon the particular plant species being transformed. The transformned cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in this way. Reference may be made to the literature for full details of the known methods.
Christou and Heie (1997) describe the transformation of rice using bombardment methodology and progress on rice transformation mediated by Agrobacterium tumefaciens. 
Other published methods for transforming wheat include Becker et al (1994) which describes the use of microprojectile bombardment of scutellar tissue and Vasil et al (1993) which describes the rapid generation of transgenic wheat following direct bombardment of immature embryos. FIG. 22 describes timelines for wheat transformation by bombardment.
The use of a selectable marker is required in the transformation process to select transformants carrying the sterility constructs. This could be an antibiotic selectable marker or a herbicide resistance gene. The use of a herbicide resistance gene or other marker is not essential (but may be considered to be convenient) to the process of hybrid seed production.
The hemizygous plants used in the second method of the present invention can be treated with chemical to induce the expression of the restorer genes which allows self pollination to occur. The progeny of this self pollination will be segregating and can be grown up, treated with chemical and self pollinated. The progeny from homozygous lines will not segregate for sterility. A repeat of the process may then be performed to bulk up homozygous sterile seed.
The individual components of the expression cassette of the present invention may be provided on one or more individual vectors. These can be used to transform or co-transform plant cells so as to allow the appropriate interaction between the elements to take place.